Two types of amino acid substitutions in protein evolution

Summary The frequency of amino acid substitutions, relative to the frequency expected by chance, decreases linearly with the increase in physico-chemical differences between amino acid pairs involved in a substitution. This correlation does not apply to abnormal human hemoglobins. Since abnormal hemoglobins mostly reflect the process of mutation rather than selection, the correlation manifest during protein evolution between substitution frequency and physico-chemical difference in amino acids can be attributed to natural selection. Outside of abnormal proteins, the correlation also does not apply to certain regions of proteins characterized by rapid rates of substitution. In these cases again, except for the largest physico-chemical differences between amino acid pairs, the substitution frequencies seem to be independent of the physico-chemical parameters. The limination of the substituents involving the largest physicochemical differences can once more be attributed to natural selection. For smaller physico-chemical differences, natural selection, if it is operating in the polypeptide regions, must be based on parameters other than those examined.     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Nonrandom amino acid substitution and estimation of the number of nucleotide substitutions in evolution

Summary A method of estimating the number of nucleotide substitutions from amino acid sequence data is developed by using Dayhoff's mutation probability matrix. This method takes into account the effect of nonrandom amino acid substitutions and gives an estimate which is similar to the value obtained by Fitch's counting method, but larger than the estimate obtained under the assumption of random substitutions (Jukes and Cantor's formula). Computer simulations based on Dayhoff's mutation probability matrix have suggested that Jukes and Holmquist's method of estimating the number of nucleotide substitutions gives an overestimate when amino acid substitution is not random and the variance of the estimate is generally very large. It is also shown that when the number of nucleotide substitutions is small, this method tends to give an overestimate even when amino acid substitution is purely at random.     

Are Radical and Conservative Substitution Rates Useful Statistics in Molecular Evolution?

A DNA mutation in a protein coding gene which causes an amino acid change can be classified as conservative or radical depending on the magnitude of the physicochemical difference between the two amino acids: radical mutations involve larger changes than conservative mutations. Here, I examine two key issues in determining whether radical and conservative substitution rates are useful statistics in molecular evolution. The first issue is whether such rates can be estimated reliably, and for this purpose I demonstrate considerable improvements achieved by simple modifications to an existing method. The second issue is whether conservative and radical substitution rates can tell us something about selection on protein function. I address this problem by estimating positive and negative selection on conservative and radical mutations using polymorphism and divergence data from Drosophila. These analyses show that negative selection, but not positive selection, differs significantly between conservative and radical mutations. The power of conservative and radical substitution rates in testing the nearly neutral theory of molecular evolution is illustrated by the analysis of two mammalian datasets.     

Percolation on fitness landscapes: effects of correlation, phenotype, and incompatibilities

We study how correlations in the random fitness assignment may affect the structure of fitness landscapes, in three classes of fitness models. The first is a phenotype space in which individuals are characterized by a large number n of continuously varying traits. In a simple model of random fitness assignment, viable phenotypes are likely to form a giant connected cluster percolating throughout the phenotype space provided the viability probability is larger than 1/2(n). The second model explicitly describes genotype-to-phenotype and phenotype-to-fitness maps, allows for neutrality at both phenotype and fitness levels, and results in a fitness landscape with tunable correlation length. Here, phenotypic neutrality and correlation between fitnesses can reduce the percolation threshold, and correlations at the point of phase transition between local and global are most conducive to the formation of the giant cluster. In the third class of models, particular combinations of alleles or values of phenotypic characters are "incompatible" in the sense that the resulting genotypes or phenotypes have zero fitness. This setting can be viewed as a generalization of the canonical Bateson-Dobzhansky-Muller model of speciation and is related to K-SAT problems, prominent in computer science. We analyze the conditions for the existence of viable genotypes, their number, as well as the structure and the number of connected clusters of viable genotypes. We show that analysis based on expected values can easily lead to wrong conclusions, especially when fitness correlations are strong. We focus on pairwise incompatibilities between diallelic loci, but we also address multiple alleles, complex incompatibilities, and continuous phenotype spaces. In the case of diallelic loci, the number of clusters is stochastically bounded and each cluster contains a very large sub-cube. Finally, we demonstrate that the discrete NK model shares some signature properties of models with high correlations.     

Evolution of the amino acid substitution in the mammalian myoglobin gene

Summary Multivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.     

Silent mutations in the gene encoding the p53 protein are preferentially located in conserved amino acid positions and splicing enhancers

The last release of p53 somatic mutation database contains more than 20,000 of mutation among which 951 are silent (synonymous). This striking amount of silent mutations is much more than what would be expected if synonymous mutations were effectively neutral. The prevalent explanation to reconcile this vast amount of silent mutations with the neutral expectation is that they are just the subproduct of the hypermutability process that affect cancer cells. Some evidences have been presented in this direction, and the explanation has been taken as granted. Assuming that silent mutations are effectively neutral has major implication in the investigation of mutational processes that affect the gene encoding the p53 protein, since on the basis of this assumption they are considered the Null hypothesis, for instance for measuring and comparing among tissues the endogenous mutability. From this it follows that determining whether silent mutations in the p53 gene, and in all disease genes in general, are or not basically mutational noise, is of paramount importance.

In this paper we readdress this topic by testing whether there is a relationship between the spatial distribution of silent mutations inside the p53 gene and functional significant features of the gene. For this purpose we divided the population of silent mutations in three groups: those that are found accompanied by other mutations (doublets and multiplest), those that were isolated as singlets, but the same mutation was also isolated as being part of a doublet (or multiplet) in another individual. And the last group is composed by those that were always found as singlets and never as being part of a doublet or a multiplet. This last group was expected to be enriched in functionally significant silent mutations. We found that all silent mutations, but particularly those of the last group, are preferentially located in conserved amino acid positions (i.e. functionally important amino acids) and also tend to be located inside suspected splicing enhancers. Noteworthy, this association remains even after eliminating the possible contribution of mutation hotspots. Besides, we present additional evidence in the direction that these putative splicing enhancers are real functional enhancers.     

On the relation between genetic and environmental variability in animals

Summary If a phenotypic character is under stabilizing selection, the selective disadvantage of a nonoptimal genotype will decrease exponentially to zero as the proportion of phenotypic variation that is environmental in origin -V _e /V _p - increases. Under the modified mutation-drift hypothesis of genetic polymorphism, the proportion of mutations that are effectively neutral and average heterozygosity should increase with this ratio. Invertebrates, because of their small size, fast development, and low degree of homeostasis (relative to vertebrates), are expected to show a larger environmental component of phenotypic variation than vertebrates. This may help explain why invertebrates are in general more genetically variable than vertebrates and why, when laboratory populations ofDrosophila are maintained in heterogeneous environments, genetic variability is lost less rapidly than when they are kept in constant conditions.     

Six amino acid substitutions in the carboxyl-transferase domain of the plastidic acetyl-CoA carboxylase gene are linked with resistance to herbicides in a Lolium rigidum population

The molecular basis of an acetyl-CoA carboxylase (ACCase) target-based resistant Lolium rigidum population (WLR 96) was studied here. The carboxyl-transferase domain of the plastidic ACCase gene from resistant individuals was amplified by PCR and sequenced. The DNA sequences were aligned and compared with a susceptible population. Six amino acid substitutions were identified in the resistant population. The substitution Ile-2041-Asn, known to confer resistance to ACCase-inhibiting herbicides aryloxyphenoxypropionate (APP) in Alopecurus myosuroides, was identified in most resistant plants but it is always linked with other amino acid substitutions. This was confirmed by a cleaved amplified polymorphism (CAP) marker and an allele-specific PCR. The sole amino acid substitution Ile-2041-Asn was not found in this population. It is likely this mutation evolved later among individuals already possessing the other substitutions. Three haplotypes were identified from the resistant population based on the six amino acid combinations, and two are linked with herbicide resistance in this population. The multiple amino acid substitutions including the Ile-2041-Asn form the molecular basis endowing a high degree of resistance to ACCase-inhibiting herbicides in this L. rigidum population.     

On the biochemical systematics of selected mammalian taxa: empirical comparison of qualitative and quantitative approaches in the evaluation of protein electrophoretic data

Empirical data sets of Artiodactyla (Antilocapridae, Bovidae, Cervidae, Suidae), Carnivora (Mustelidae) and Rodentia (Sciuridae, Cricetidae, Arvicolidae, Muridae), obtained by horizontal starch el electrophoresis of 15–34 isoenzyme sstems, were used to calculate genetic distances and to construct phylogenetic trees by the following methods: Nei's D (corrected for small sample sizes) - UPGMA, FITCH, KITSCH (out of Felsenstein's PHYLIP-package); Rogers -distance - distance-Wanger tree; maximum likelihood approach (cavalli -Sforza -Edwards ); maximum parsimony method (wagner ); Hennigian cladogram. The results were re-examined using the statisticar methods of jackknife and bootstrap. The following problems became apparent and were studied in more detail: inconstancy of molecular evolutionary rate among taxa, non-uniformity of evolutionary rate among isoenzymes, possible convergence of alloenzymes, different evolutionary histories of taxa (radiations/bottlenecks), methodological influences sample sizes / rare alleles, comparability of data sets). The results show, that many branches of the various phylogenetic trees are fairly constant. The ambiguous position of the remaining OTU's is due to insufficient evidence in the primary data rather than to theroperties of cluster algorithms. However, since these problematic cases are also uncertain in phylogenies based on morphological characters and palaeontological results, even an increased data set may not lead to a cyear decision unless additional taxa of crucial importance are examined. Molecular evolutionary rate among taxa seems to be accelerated in some cases, possibly due to random fixation of different alleles during bottlenecks, when a highly polymorpic ancestral form underwent a series of adaptive radiations. Isoenzymes can be divided into groups with different evolutionary rates. Thus, data sets are only comparable with respect to genetic variability and differentiation, when they contain a similar amount of representatives of each of these categories.     

Molecular evolution of mRNA: A method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application

Summary A method for estimating the evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences is presented. This method is applied to genes of øX174 and G4 genomes, histone genes and-globin genes, for which homologous nucleotide sequences are available for comparison to be made. It is shown that the rates of synonymous substitutions are quite uniform among the non-overlapping genes of øX174 and G4 and among histone genes H4, H2B, H3 and H2A. A comparison between øX174 and G4 reveals that, in the overlapping segments of the A-gene, the rate of synonymous substitution is reduced more significantly than the rate of amino acid substitution relative to the corresponding rate in the nonoverlapping segment. It is also suggested that, in the coding regions surrounding the splicing points of intervening sequences of-globin genes, there exist rigid secondary structures. It is in only these regions that the-globin genes show the slowing down of evolutionary rates of both synonymous and amino acid substitutions in the primate line.     

The evolution of proteins from random amino acid sequences: II. Evidence from the statistical distributions of the lengths of modern protein sequences

This paper continues an examination of the hypothesis that modern proteins evolved from random heteropeptide sequences. In support of the hypothesis, White and Jacobs (1993, J Mol Evol 36:79–95) have shown that any sequence chosen randomly from a large collection of nonhomologous proteins has a 90% or better chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. The goal of the present study was to investigate the possibility that the random-origin hypothesis could explain the lengths of modern protein sequences without invoking specific mechanisms such as gene duplication or exon splicing. The sets of sequences examined were taken from the 1989 PIR database and consisted of 1,792 super-family proteins selected to have little sequence identity, 623 E. coli sequences, and 398 human sequences. The length distributions of the proteins could be described with high significance by either of two closely related probability density functions: The gamma distribution with parameter 2 or the distribution for the sum of two exponential random independent variables. A simple theory for the distributions was developed which assumes that (1) protoprotein sequences had exponentially distributed random independent lengths, (2) the length dependence of protein stability determined which of these protoproteins could fold into compact primitive proteins and thereby attain the potential for biochemical activity, (3) the useful protein sequences were preserved by the primitive genome, and (4) the resulting distribution of sequence lengths is reflected by modern proteins. The theory successfully predicts the two observed distributions which can be distinguished by the functional form of the dependence of protein stability on length.The theory leads to three interesting conclusions. First, it predicts that a tetra-nucleotide was the signal for primitive translation termination. This prediction is entirely consistent with the observations of Brown et al. (1990a,b, Nucleic Acids Res 18:2079–2086 and 18: 6339-6345) which show that tetra-nucleotides (stop codon plus following nucleotide) are the actual signals for termination of translation in both prokaryotes and eukaryotes. Second, the strong dependence of statistical length distributions on sequence-termination signaling codes implies that the evolution of stop codons and translation-termination processes was as important as gene splicing in early evolution. Third, because the theory is based upon a simple no-exon stochastic model, it provides a plausible alternative to a limited universe of exons from which all proteins evolved by gene duplication and exon splicing (Dorit et al. 1990, Science 250:1377–1382).     

The role of the conserved Lys68*:Glu265 intersubunit salt bridge in aspartate aminotransferase kinetics: multiple forced covariant amino acid substitutions in natural variants

The role of the Lys68*:Glu265 intersubunit salt bridge that is conserved (Csb) in all known aspartate aminotransferases (AATases), except those of animal cytosolic, Ac (His68*:Glu265), and plant mitochondrial, Pm (Met68*:Gln265), origins, was evaluated in the Escherichia coli AATase. Two double-mutant cycles, to K68M/E265Q and the charge reversed K68E/E265K, were characterized with the context dependence (C) and impact (I) formalism, previously defined for functional chimeric analysis. Mutations of Lys68* with Glu265 fixed are generally more deleterious than the converse mutations of Glu265 with Lys68* fixed, showing that buried negative charges have greater effects than buried positive charges in this context. Replacement of the charged Lys68*:Glu265 with the K68M/E265Q neutral pair introduces relatively small effects on the kinetic parameters. The differential sensitivity of k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) to salt bridge mutagenic replacements is shown by a linear-free energy relationship, in which the logarithms of the latter second order rate constants are generally decreased by a factor of two more than are those of the former. Thus, k(cat)/K(M, L-Asp) and k(cat)/K(M, alpha-KG) are 133 and 442 mM(-1)s(-1) for the wild-type (WT) enzyme, respectively, but their relative order is reversed in the more severely compromised mutants (14.8 and 5.3 mM(-1)s(-1) for K68E). A Venn diagram illustrates apparent forced covariances of groups of amino acids that accompany the naturally occurring salt bridge replacements in the Pm and Ac classes. The more deeply rooted tree indicates that the Csb variant was the ancestral specie.     

On the appearance and role of a spacer group in the protein amino acids

Summary Of the 20 protein amino acids, 16 have a methylene group at the position, and a further three bear a methine group. No aromatic, carboxamido, carboxylic carbon, or hetero atoms are attached directly to the carbon, but they are separated by this methylene or occasionally by a longern-alkylene spacer group. Therefore, the structure of the protein amino acids should rather be formulated as H₂N–CH((CH₂)_n–R)–COOH instead of the generally accepted H₂N–CH(R)–COOH. The appearance of and the role played by the spacer group are discussed in an evolutionary context. It is suggested that the spacer group appeared as a result of prebiotic selection, based on the relative abundance, racemization rate, and suitability for thermal polymerization of the protein amino acids and their homologs with various spacer group lengths. At the biotic level of evolution the requirements for ribosomal polymerization, as well as the abilities of polypeptides to maintain a stable and flexible threedimensional structure and to bind ligands are considered and are proposed to have been responsible for the possible exclusion of longer spacer groups. It is concluded that the general role of the spacer group is to ensure the uniformity of the constant regions H₂N–CH(-)–COOH and the individuality of the R contact groups by spatially separating them.